Unraveling molecular effects of ADAR1 overexpression in HEK293T cells by label-free quantitative proteomics

ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up- and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up- and 159 are down-regulated for at least 1.5-fold (n = 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.     

Quantitative proteomics reveals GIMAP family proteins 1 and 4 to be differentially regulated during human T helper cell differentiation

T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4(+) cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation.     

Proteome profiling of interleukin-12 treated human T helper cells

Selective activation of T helper subsets 1 (Th1) and 2 (Th2) plays a crucial role in different pathological conditions. Th1 cell response is involved in pathogenesis of autoimmune diseases, such as type II diabetes and multiple sclerosis, and Th2 cell response in pathogenesis of allergy and asthma. Cytokine interleukin-12 (IL-12) is one of the key factors in the differentiation of na?ve CD4(+) T cells into Th1 cells. In this study we used 2-DE and MS to find and identify IL-12 regulated proteins in human CD4(+) T cells. In total, 42 protein spots were found to be differentially expressed following IL-12 stimulation, of which 22 were up- and 20 down-regulated. Among the upregulated proteins there are a multifunctional cytokine macrophage migration inhibitory factor and a known IL-12 target gene Programmed cell death 4. Downregulated proteins include p21-activated kinase 2 and its upstream GTPase Cdc42. Compared to previous reports our analysis provides a new view on the IL-12 induced changes on CD4(+) T cells underscoring the importance of creating and combining the data generated at various levels to build a comprehensive view of a given biological process of the cell.     

Comparative kinetic analyses of gene profiles of naïve CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naïve CD4⁺ and CD8⁺ T cells, microarray analysis was performed pre‐ and post‐TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T‐cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4⁺ and CD8⁺ naïve T cells. Evaluation of these differences provides in‐depth insight into potential functional and phenotypical differences among these populations.     

Systems biology and proteomic analysis of cerebral cavernous malformation

Cerebral cavernous malformations (CCM) are vascular anomalies caused by mutations in genes encoding KRIT1, OSM and PDCD10 proteins causing hemorrhagic stroke. We examine proteomic change of loss of CCM gene expression. Using human umbilical vein endothelial cells, label-free differential protein expression analysis with multidimensional liquid chromatography/tandem mass spectrometry was applied to three CCM protein knockdown cell lines and two control cell lines: ProteomeXchange identifier PXD000362. Principle component and cluster analyses were used to examine the differentially expressed proteins associated with CCM. The results from the five cell lines revealed 290 and 192 differentially expressed proteins (p < 0.005 and p < 0.001, respectively). Most commonly affected proteins were cytoskeleton-associated proteins, in particular myosin-9. Canonical genetic pathway analysis suggests that CCM may be a result of defective cell–cell interaction through dysregulation of cytoskeletal associated proteins. Conclusion: The work explores signaling pathways that may elucidate early detection and novel therapy for CCM.     

Single‐cell multi‐omics analysis presents the landscape of peripheral blood T‐cell subsets in human chronic prostatitis/chronic pelvic pain syndrome

Cumulative evidence suggests that abnormal differentiation of T lymphocytes influences the pathogenesis of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Thus, understanding the immune activation landscape of CP/CPPS would be helpful for improving therapeutic strategies. Here, we utilized BD™ AbSeq to digitally quantify both the protein and mRNA expression levels in single peripheral blood T cells from two CP/CPPS patients and two healthy controls. We utilized an integrated strategy based on canonical correlation analysis of 10 000+ AbSeq profiles and identified fifteen unique T‐cell subpopulations. Notably, we found that the proportion of cluster 0 in the CP/CPPS group (30.35%) was significantly increased compared with the proportion in the healthy control group (9.38%); cluster 0 was defined as effector T cells based on differentially expressed genes/proteins. Flow cytometry assays confirmed that the proportions of effector T‐cell subpopulations, particularly central memory T cells, T helper (Th)1, Th17 and Th22 cells, in the peripheral blood mononuclear cell populations of patients with CP/CPPS were significantly increased compared with those of healthy controls (P < 0.05), further confirming that aberration of effector T cells possibly leads to or intensifies CP/CPPS. Our results provide novel insights into the underlying mechanisms of CP/CPPS, which will be beneficial for its treatment.     

Proteomic analysis of rosiglitazone and guggulsterone treated 3T3-L1 preadipocytes

Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5–7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone.     

Identification of key candidate genes and pathways in multiple myeloma by integrated bioinformatics analysis

Multiple myeloma (MM) is a common hematologic malignancy for which the underlying molecular mechanisms remain largely unclear. This study aimed to elucidate key candidate genes and pathways in MM by integrated bioinformatics analysis. Expression profiles GSE6477 and GSE47552 were obtained from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) with p < .05 and [logFC] > 1 were identified. Functional enrichment, protein–protein interaction network construction and survival analyses were then performed. First, 51 upregulated and 78 downregulated DEGs shared between the two GSE datasets were identified. Second, functional enrichment analysis showed that these DEGs are mainly involved in the B cell receptor signaling pathway, hematopoietic cell lineage, and NF-kappa B pathway. Moreover, interrelation analysis of immune system processes showed enrichment of the downregulated DEGs mainly in B cell differentiation, positive regulation of monocyte chemotaxis and positive regulation of T cell proliferation. Finally, the correlation between DEG expression and survival in MM was evaluated using the PrognoScan database. In conclusion, we identified key candidate genes that affect the outcomes of patients with MM, and these genes might serve as potential therapeutic targets.     

CD226 is specifically expressed on the surface of Th1 cells and regulates their expansion and effector functions

Surface molecules that are differentially expressed on Th1 and Th2 cells may be useful in regulating specific immune responses in vivo. Using a panel of mAbs, we have identified murine CD226 as specifically expressed on the surface of differentiated Th1 cells but not Th2 or Th0 cells. Although CD226 is constitutively expressed on CD8 cells, it is up-regulated on CD4 cells upon activation. Th1 differentiation results in enhanced CD226 expression, whereas expression is down-regulated upon Th2 polarization. We demonstrate that CD226 is involved in the regulation of T cell activation; in vivo treatment with anti-CD226 results in significant reduction of Th1 cell expansion and in the induction of APCs that inhibit T cell activation. Furthermore, anti-CD226 treatment delays the onset and reduces the severity of a Th1-mediated autoimmune disease, experimental autoimmune encephalomyelitis. Our data suggest that CD226 is a costimulatory molecule that plays an important role in activation and effector functions of Th1 cells.     

SOCS proteins in T helper cell differentiation: implications for allergic disorders?

Asthma, allergic rhinitis and atopic dermatitis are allergic immune disorders characterised by a predominance of T helper 2 (Th2) cells, the resulting elevation of allergen-specific IgE, and mast-cell- and basophil-associated inflammation. The cytokine environment at the site of the initial antigen stimulation determines the direction of Th-cell differentiation into Th1 or Th2 cells. The SOCS (suppressor of cytokine signalling) proteins are implicated in the control of the balance between Th1 and Th2 cells in this process. SOCS3 is predominantly expressed in Th2 cells and inhibits Th1 differentiation; conversely, SOCS5 is expressed predominantly in Th1 cells and inhibits Th2 differentiation. Here, we discuss the role of SOCS proteins in Th-cell differentiation and explore the potential of SOCS proteins as targets for therapeutic strategies in allergic disorders.     

Characterization of Differentially Expressed Genes Involved in Pathways Associated with Gastric Cancer

To explore the patterns of gene expression in gastric cancer, a total of 26 paired gastric cancer and noncancerous tissues from patients were enrolled for gene expression microarray analyses. Limma methods were applied to analyze the data, and genes were considered to be significantly differentially expressed if the False Discovery Rate (FDR) value was < 0.01, P-value was <0.01 and the fold change (FC) was >2. Subsequently, Gene Ontology (GO) categories were used to analyze the main functions of the differentially expressed genes. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we found pathways significantly associated with the differential genes. Gene-Act network and co-expression network were built respectively based on the relationships among the genes, proteins and compounds in the database. 2371 mRNAs and 350 lncRNAs considered as significantly differentially expressed genes were selected for the further analysis. The GO categories, pathway analyses and the Gene-Act network showed a consistent result that up-regulated genes were responsible for tumorigenesis, migration, angiogenesis and microenvironment formation, while down-regulated genes were involved in metabolism. These results of this study provide some novel findings on coding RNAs, lncRNAs, pathways and the co-expression network in gastric cancer which will be useful to guide further investigation and target therapy for this disease.     

Effector CD4+ T cell expression signatures and immune-mediated disease associated genes

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in na?ve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to na?ve CD4+ T cells of genes contained among immune-mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.     

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.     

Differential expression of Dicer and Argonaute genes duringthe differentiation of human neuroblastoma cells

Dicer and Argonaute 1-4 proteins are key components of the cytoplasmic enzyme machinery responsible for biogenesis and performance of microRNAs. To gain insight into the roles of these proteins in cell differentiation, we investigated possible changes in the expression levels of Dicer and Argonaute 1-4 genes during the differentiation of cultured neural and glial cells. The results show that the 5 genes are differentially expressed along the 2 differentiation pathways and suggest a prevalent role of Dicer and Argonaute 4 in neural cell differentiation.     

Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.